Controlling Topology and Native-like Behavior of de Novo-Designed Peptides:  Design and Characterization of Antiparallel Four-Stranded Coiled Coils

Abstract

The de novo design of peptides and proteins has emerged as an attractive approach for investigating protein structure and function. Here, the design, synthesis, and characterization of a new series of alpha-helical peptides intended to form antiparallel four-stranded coiled coils is described. Computer models were generated without the use of extant protein structures and were used to refine the sequence. The peptides are of the general formula Ncap-(XaZbZcLdZeZfZg)3-Ccap, where X is either Ala, Val, Thr, or Leu, and Ncap and Ccap are sequences designed to satisfy the helices unpaired amide nitrogens and carbonyl oxygens, respectively. The hydrophobic residues (at positions a and d) were chosen so that geometric packing of large and small hydrophobes would favor an antiparallel arrangement. Special attention was also given to residues at the helix--helix interfaces. These residues were chosen to balance potential attractive and repulsive electrostatic forces so that the desired topology was favored while other possible folds were destabilized. Two of the four peptides associate under neutral conditions into the desired tetramers. One of the complexes (a = Val) behaves like a native-like protein as judged by NMR, thermodynamics, and apolar dye (ANS) binding. The other tetrameric complex (a = Leu) exhibits broader NMR resonances, diminished values of delta H and delta Cp, and tight binding of the hydrophobic dye ANS, similar to early designed proteins. These results reinforce the importance of optimizing van der Waals packing interactions in protein design but demonstrate that hydrophobic packing must be balanced with hydrogen-bonding and electrostatic interactions to produce novel native-like proteins.

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

“…Our retrostructural analysis suggests that many complex metalloproteins may have evolved from primordial precursors that were formed by the noncovalent self assembly of simple secondary-structure motifs (59,79,80). Covalent connection of the individual secondary structures may have occurred at a later date, via either random shuffling of nonidentical units to give asymmetric proteins or gene duplication to give a symmetric protein.…”

“…Often the metal ionbinding sites of metalloproteins are formed between two or more elements of secondary structure, a fact that allowed the correct prediction of the fold of the zinc finger before its experimental determination (59). A retrostructural approach can be used to identify geometric relationships between these secondary structural elements.…”

Coupling backbone flexibility and amino acid sequence selection in protein design

“…Several groups have proposed and tested systematic, quantitative methods for protein design that screen possible sequences for compatibility with a desired backbone fold (Ponder & Richards, 1987;Hellinga et al, 1991;Hurley et al, 1992;Hellinga & Richards, 1994;Desjarlais & Handel, 1995;Harbury et al, 1995;Klemba et al, 1995;Nautiyal et al, 1995;Betz & Degrado, 1996;Dahiyat & Mayo, 1996). In these methods, the backbone is held fixed and a search is performed to find side chains, whose conformations are often discretized as rotamers, that lead to sterically acceptable packing arrangements.…”

Automated design of the surface positions of protein helices

“…The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 "C, with melting temperatures from 69-72 "C, over 12 "C higher than GCNCpl, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 "C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.Keywords: coiled coils; helix propensities; protein design; surface residuesSeveral groups have proposed and tested systematic, quantitative methods for protein design that screen possible sequences for compatibility with the desired protein fold (Hellinga et al, 1991;Hurley et al, 1992;Desjarlais & Handel, 1995;Harbury et al, 1995;Klemba et al, 1995;Nautiyal et al, 1995;Betz & Degrado, 1996;Dahiyat & Mayo, 1996). These algorithms consider the spatial positioning and steric complementarity of side chains by explicitly modeling the atoms of sequences under consideration.…”

“…Several groups have proposed and tested systematic, quantitative methods for protein design that screen possible sequences for compatibility with the desired protein fold (Hellinga et al, 1991;Hurley et al, 1992;Desjarlais & Handel, 1995;Harbury et al, 1995;Klemba et al, 1995;Nautiyal et al, 1995;Betz & Degrado, 1996;Dahiyat & Mayo, 1996). These algorithms consider the spatial positioning and steric complementarity of side chains by explicitly modeling the atoms of sequences under consideration.…”